In the course of studying inflammatory muscle diseases (polymyositis, dermatomyositis, and related diseases), we have encountered patients with many other muscle diseases. We have studied patients with two genetic metabolic myopathies in detail: phosphofructokinase (PFK) deficiency, and acid maltase (acid alpha-glucosidase) deficiency (also known as Glycogen Deficiency Type II or GSD II or Pompe Syndrome). For the last several years, we have focused particular attention on GSD II because of its close resemblance to myositis. It is a recessively inherited lysosomal storage disease in which glycogen accumulates in the lysosomes, particularly those in skeletal muscle. When the enzyme, known also as GAA, is completely absent, affected infants are usually sick at birth and die in infancy of heart failure, rarely living longer than a year. Apparently the enzyme is needed in the heart only in infancy since affected individuals with even a small amount of effective enzyme survive without cardiac involvement. Survivors generally develop a progressive proximal myopathy with pulmonary failure secondary to diaphragmatic involvement in later years. In those with about 10% of normal levels of GAA, the disease presents in adult life with a symmetric proximal myopathy often accompanied by pulmonary failure that may be indistinguishable from polymyositis. Such patients are often treated with ineffective and potentially harmful immunosuppressive drugs. The long-term aim of our studies is to prevent and to treat this devastating disease, particularly the adult variety, in which the level of enzyme is only slightly (less than two-fold) below the minimum necessary for a normal life. Since much of the research effort in the rest of the world has been driven by pediatricians faced with the fatal infantile disease, we have been drawn into collaborations with them. Work in our own laboratory has been in two major areas: understanding the transcriptional control of the enzyme, and developing an animal models for testing recombinant protein and gene replacement therapies. Earlier studies in our laboratory have shown that adults produce low levels of acid maltase that is structurally normal owing to an intronic mutation that slows splicing of one intron. We also identified a silencer elsewhere in this intron, and thus turned our attention to understanding this silencer in detail in order to try to accelerate transcription in adult patients. We had localized the silencer activity in Hep G2 cells to a 25 bp region within which there are adjacent sites for two known transcription factors, YY1 and Hes-1. Experiments have shown that both of these factors bind to the region. By mutating the region, it has further been shown that alteration of either site is sufficient to reverse the silencer activity. These studies establish, therefore, that in this region there is a control point at which two transcription factors work cooperatively to down-regulate transcription of the gene. The findings so far are intrinsically interesting since so little is known of the control of so-called housekeeping genes. In fact, this is the first example of transcriptional control of a gene in this family localized to an intron. The continuation of these studies in fibroblasts has shown that in this cell type, the very same region functions as an enhancer rather than as a silencer despite the fact that the same factors are involved. Of particular interest is the involvement of Hes-1, a target gene in the Notch signaling pathway, in the control of GAA. Hes-1 is a gene that is critical for nervous system and thymic maturation, known previously only as a silencer. The recent studies have now clearly shown that Hes-1 is one of the few transcription factors that can function as either a silencer or an enhancer. Furthermore, recent experiments have established that Notch, acting through Hes-1, is connected to the transcription of this housekeeping, lysosomal enzyme. Related studies with fibroblasts have shown that another region and factors in intron 1 have silencing activity, thus establishing the tissue-specific nature of the transcriptional control of GAA. With the impending departure of Dr. Bo Yan for George Washington University, the future of this line of research has become uncertain. In order to provide an animal model for testing several proposed therapies for acid maltase deficiency which are actively under development in our group and by groups in the Netherlands, in New York, at Duke, and at Johns Hopkins, we had made knockout models of the GAA gene in mice. Homozygous F2 offspring rapidly accumulate glycogen in cardiac and skeletal muscle. Females show impaired performance in muscle testing by quantitative open field observation and by ability to hang on a wire screen or move on a rotating rod, and by several months, they develop a grossly waddling gait. The exon 6 knockout was designed so that the neo gene and the whole of exon 6 could be deleted permanently by mating the mice to mice transgenic for the CRE recombinase. These mice have a slower onset of clinical disease despite total absence of GAA activity, because of differences in the background genes of the two strains. In addition, in the course of experiments to accelerate the onset of clinical disease in knockout mice by increasing glycogen synthesis, we discovered that mice with transgenic glycogen synthetase accumulated not more glycogen but another glucose polymer, polyglucosan, thus accidentally pointing the way to an animal model of another glycogen storage disease, Brancher disease, and its CNS relative, polyglucosan body disease, a common childhood epilepsy due to the abnormal storage of polyglucosan. This knowledge will allow development of an experimental animal model of that disease. In the past year, our efforts have been directed at studying the factors that must be understood in order to replace GAA by providing recombinant human enzyme or by gene therapy in affected patients by studying those factors in our knockout mice. We have placed a transgene that can express GAA in knockout mice under the control of a liver-specific or a muscle-specific promoter. The promoters are tetracycline-controllable, thus allowing us to ask a number of useful questions. Following the complex manipulations to develop the relevant strains, we have in hand lines that make biologically effective enzyme at low, medium, or high levels in the liver or the skeletal muscle, and one line that makes it only in the heart. We have established that the liver proved a far more efficient source of enzyme than does skeletal muscle. A large quantity of biologically active enzyme is secreted by the liver. The achievement of therapeutic levels with skeletal muscle transduction required the entire muscle mass comprising 40 % of body weight to provide high levels of enzyme of which little found its way to the plasma, whereas liver, comprising less than 5% of body weight, secreted 110-fold more enzyme all of which was in the active 110 kDa precursor form. Using tetracycline regulation, we somatically induced human GAA in the knockout mice and demonstrated that the skeletal and cardiac muscle pathology was completely reversible. If, however, the enzyme is turned on late in the illness, reversibility has been less complete in on-going experiments. These observations have important implications for those planning enzyme replacement therapy. Our collaborators in the Department of Pediatrics at Duke have established that enzyme made by an adenoviral vector that has settled in the liver is secreted in sufficient quantity to reverse glycogen accumulation in muscle of our knockout mice. Our collaborators in the Department of Pediatrics at Johns Hopkins and at the University of Florida have developed an adeno-associated virus (AAV) vector with the acid maltase gene.